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Problem with cloning RAPD fragment

Eugene Tanimoto yuji at U.WASHINGTON.EDU
Tue Jan 19 18:05:47 EST 1993

Dear Yin-Chai,

	I've been cloning gel isolated RAPD fragments with Novagen's
pT7Blue vector (800-526-7319).  It's similar to other T-vectors like
Invitrogen and others and allows blue-white selection.  I've cloned
fragments up to 1000 bp.  Generally, I've isolated the RAPD band of
interest by electro-elution of the agarose-frag, ethanol ppt, and use the
resuspended fragment as template for a re-PCR of the RAPD fragment using
the same primer.  I've checked a couple of the cloned inserts by using
them as a probe for a Southern blot of the RAPD fragments from the
original amplification and they seem to light up the correct band. Novagen
recommends using 50 ng of vector in the ligation with the insert.  I've
transformed DH5 alpha that were only 10 to the sixth power competent and
get on the average, 200 colonies that are half white, half blue (few light
blue).  I pick only the white colonies to screen and most contain the
proper size insert.  I hope this helps.  Gene Tanimoto (yuji at u.washington.edu)

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